PtWRKY2, a WRKY transcription factor from Pinellia ternata confers heat tolerance in Arabidopsis

High temperatures are a major stress factor that limit the growth of Pinellia ternata. WRKY proteins widely distribute in plants with the important roles in plant growth and stress responses. However, WRKY genes have not been identified in P. ternata thus far. In this study, five PtWRKYs with four functional subgroups were identified in P. ternata. One group III WRKY transcription factor, PtWRKY2, was strongly induced by high temperatures, whereas the other four PtWRKYs were suppressed. Analysis of transcription factor characteristics revealed that PtWRKY2 localized to the nucleus and specifically bound to W-box elements without transcriptional activation activity. Overexpression of PtWRKY2 increased the heat tolerance of Arabidopsis, as shown by the higher percentage of seed germination and survival rate, and the longer root length of transgenic lines under high temperatures compared to the wild-type. Moreover, PtWRKY2 overexpression significantly decreased reactive oxygen species accumulation by increasing the catalase, superoxide dismutase, and peroxidase activities. Furthermore, the selected heat shock-associated genes, including five transcription factors (HSFA1A, HSFA7A, bZIP28, DREB2A, and DREB2B), two heat shock proteins (HSP70 and HSP17.4), and three antioxidant enzymes (POD34, CAT1, and SOD1), were all upregulated in transgenic Arabidopsis. The study identifies that PtWRKY2 functions as a key transcriptional regulator in the heat tolerance of P. ternata, which might provide new insights into the genetic improvement of P. ternata.

www.nature.com/scientificreports/Pinellia ternata is a herb belonging to the Araceae family that is widely distributed in China and Southeast Asia.Its tubers contain alkaloids, organic acids, and polysaccharides 14,15 , and are used for medicinal purposes.Modern pharmacological studies have shown that P. ternata has many medicinal properties, including analgesic, anxiolytic, antitussive, and anticancer effects 16 .The suitable temperature for the growth of P. ternata is 15-25 °C.When exposed to high temperatures during growth, P. ternata is susceptible to withering, a phenomenon known as "sprout tumble" (ST) 17,18 .ST formation in P. ternata shortens its growth period, which is a key limiting factor in tuber production 19 .Therefore, it is important to analyze the ST mechanism and delay the process of ST for improving the yield of P. ternata .Thus far, only the functions of PtSAD and PtsHSP in P. ternata in response to high temperatures have been reported 20,21 , and the transcriptional regulatory network of P. ternata related to ST is still largely unknown.
In this study, we isolated WRKY genes from P. ternata based on the full-length transcriptome and identified a WRKY gene, PtWRKY2, that was significantly induced by high temperatures.Moreover, the function of PtWRKY2 in high-temperature tolerance was investigated.These data provide new insights into ST mechanisms at the transcriptional level, which could contribute to the genetic improvement of P. ternata.
Phylogenetic analysis indicated that the 25 WRKYs from P. ternata, Oryza sativa, and Arabidopsis could be divided into six subclasses.PtWRKY1, AtWRKY25, AtWRKY26, and AtWRKY33 clustered into Group I, whereas PtWRKY2, together with the closely related AtWRKY30, AtWRKY53, and OsWRKY72, were categorized as Group III.PtWRKY3 and PtWRKY4, together with the closest AtWRKY6, belonged to Group IIb and PtWRKY5 clustered with Group IId (Fig. 1).

Expression pattern analysis of PtWRKY genes
The expression profiles of the five PtWRKY genes were investigated using quantitative real-time PCR.The five PtWRKY genes existed in all tissues of P. ternata, with PtWRKY1, PtWRKY3, PtWRKY4, and PtWRKY5 highly expressed in the roots, and PtWRKY2 highly expressed in the leaves (Fig. 2A).In terms of their heat responses, we observed that only the PtWRKY2 transcription level significantly increased within 24 h of treatment, with an expression peak at 12 h (nearly 200-fold induction).However, the expression levels of the other four PtWRKY www.nature.com/scientificreports/genes significantly decreased under stress treatment (Fig. 2B).These results implied that the induction of PtWRKY2 might participate in the growth regulation of P. ternata at high temperatures.

Transcription factor characteristics of PtWRKY2
To identify the biological functions of PtWRKY2 in response to high temperatures, PtWRKY2 was selected for further study.First, the PtWRKY2-GFP and GFP vectors were extracted for subcellular localization analysis.The cells were then transformed into tobacco epidermal cells for observation under a fluorescence microscope.As shown in Fig. 3A, the PtWRKY2-GFP fusion proteins were found only in the cell nucleus, whereas the control GFP signals were widely distributed throughout the cell, indicating that PtWRKY2 encodes a nuclear protein.
Second, to confirm the transcriptional activity of PtWRKY2, the plasmids pGBKT7-PtWRKY2, pGBKT7-53 (positive control), and pGBKT7 (negative control) were used for a transcriptional activation assay in Y2HGold yeast cells.The results revealed that all yeast cells transformed with the three vectors grew normally on SD/-Trp plates.Only the positive control grew on the SD/-Trp/-His/-Ade plate, and turned blue with the addition of X-α-gal, whereas the transformants containing pGBKT7-PtWRKY2 and pGBKT7 both failed to grow (Fig. 3B), which suggests that the PtWRKY2 protein did not have transcriptional activation activity.
WRKY transcription factors regulate gene expression by specifically binding to W-box elements within the target gene promoter 22 .To further explore whether PtWRKY2 could bind to W-box, the plasmid PtWRKY2-62-SK was grouped with W-box-0800, mW-box-0800, and pGreenII-0800, respectively.The three combined plasmids were injected into tobacco epidermal cells for the dual-luciferase reporter assay.As shown in Fig. 3C, LUC luminescence signals were produced only when the PtWRKY2-62-SK and W-box-0800 vectors were cotransformed.These results suggest that PtWRKY2 is a typical WRKY protein that specifically binds to W-box elements.

Overexpression of PtWRKY2 enhanced the heat tolerance of transgenic Arabidopsis
To confirm the function of PtWRKY2, we obtained PtWRKY2-overexpressing lines in Arabidopsis (OE1-2, OE6-3, and OE7-8) (Fig. 4A), which were used for further analyses in combination with WT Arabidopsis.In the seed germination assay, the germination rates of the WT and transgenic lines reached 100% under normal conditions.When exposed to heat stress for two days (2DHS), the germination rate exhibited a downward trend, with a > 90% rate in the transgenic lines and a 73.6%rate in the WT lines.After 3DHS treatment, the germination rate of OE6-3 reached 48.1%, followed by 36.2% in OE7-8, and 31.5% in OE1-2, while only 12.8% of the WT seeds germinated (Fig. 4B-D).The effects of PtWRKY2 on root length were also evaluated using the above Arabidopsis lines.As shown in Fig. 4E,F, the root length of the transgenic lines was greater than that of the WT plants under normal conditions, and root growth was inhibited under high temperature conditions.Notably, root length inhibition was less in the transgenic lines than in the WT plants.
The heat tolerance of the transgenic and WT Arabidopsis plants was further evaluated by exposure to heat stress.After 1DHS, the survival rate of the WT line plants was only 31.8%, whereas the survival rates of the OE1-2, OE6-3, and OE7-8 lines were 76.9%, 80.3%, and 81.9%, respectively (Fig. 5).These results indicate that the overexpression of PtWRKY2 in Arabidopsis could significantly enhance heat tolerance.It is well known that plant senescence induced by abiotic stress is usually associated with the accumulation of reactive oxygen species (ROS), particularly H 2 O 2 and O 2 -.The levels of H 2 O 2 and O 2 -in the leaves were detected by DAB and NBT staining.The PtWRKY2-overexpressing lines exhibited lighter histochemical staining than the WT leaves under heat stress conditions, whereas no significant difference was observed under control conditions (Fig. 6A,B).The activities of PtCAT, PtSOD, and PtPOD were determined in transgenic and WT Arabidopsis.The results revealed that the PtCAT, PtSOD, and PtPOD activities were similar in the WT and transgenic Arabidopsis under normal conditions.When exposed to high temperatures, the activities increased in both WT and transgenic lines; however, the increment in transgenic plants was higher than that in WT plants (Fig. 6).These results suggest that PtWRKY2 induces the activities of PtCAT, PtSOD, and PtPOD, thereby reducing ROS accumulation in transgenic Arabidopsis.

Overexpression of PtWRKY2 altered the transcriptional expression levels of heat shock associated genes
Because the overexpression of PtWRKY2 enhanced the high-temperature tolerance of Arabidopsis, the expression profiles of heat shock-associated genes were analyzed in the WT and transgenic lines under high-temperature conditions.The expression levels of five heat shock-related transcription factor-encoding genes, HSFA1A, HSFA7A, bZIP28, DREB2A, and DREB2B, were significantly higher in the PtWRKY2 overexpressed lines compared to that in the WT plants.Moreover, three genes related to antioxidant protection (POD34, CAT1, and SOD1) were upregulated in the transgenic lines.In addition, the overexpression of PtWRKY2 increased the transcript levels of the heat shock protein-coding genes HSP70 and HSP17.4 (Fig. 7).These results suggest that PtWRKY2 enhances the expression of heat shock-associated genes, thereby conferring heat tolerance to plants.

Discussion
WRKY transcription factors are widely distributed in plants and have been identified in many species, including Arabidopsis 23 , rice 24 , and wheat 25 , etc. WRKY proteins play an important regulatory role in many life processes and stress responses, and act as an important part of the plant stress signal transduction pathway [26][27][28] .In the present study, we identified five PtWRKY genes and analyzed the function of PtWRKY2 in response to heat www.nature.com/scientificreports/stress, which could be helpful in understanding the transcriptional regulation of ST in P. ternata under hightemperature stress.WRKY expression is usually tissue specific and is often affected by stress, thereby playing a role in regulating various biological processes in plants.In peppers, CaWRKY40 is induced by heat stress and functions as a positive regulator of heat stress tolerance 29 .In potatoes, the leaf-specific genes StWRKY016, StWRKY045, and StWRKY055 act as regulators of heat stress 30 .In accordance with these reports, PtWRKY2 was highly expressed in the leaves of P. ternata and significantly upregulated at high temperatures.Furthermore, PtWRKY2 is closely related to AtWRKY30, which enhances thermotolerance in wheat 31 , which suggests that PtWRKY2 most likely plays an important role for P. ternata in terms of the response to heat stress.In addition to heat stress-related WRKY genes in Arabidopsis 13 , WRKYs have been shown to function in high temperature tolerance in other species.For example, overexpression of OsWRKY11 increases the heat resistance of rice, whereas TaWRKY1 and TaWRKY33 confer heat tolerance in Arabidopsis 32,33 .Here, the overexpression of PtWRKY2 in Arabidopsis increased seed germination, root elongation, and seedling survival at high temperatures, suggesting that PtWRKY2 positively regulates thermotolerance.Basing on the phylogenetic analysis, PtWRKY2 is also grouped together with AtWRKY53 and OsWRKY72, which is related with the drought stress signal 34,35 .Thereby, it is speculated that PtWRKY2 probably also functions in the drought stress of P. ternata.
Heat stress often leads to the excessive accumulation of ROS in plants, causing oxidative damage and leading to plant senescence 36 .In P. ternata, ROS accumulation and antioxidant enzyme activity fluctuate under heat stress 37,38 , suggesting that dynamic ROS accumulation is closely related to senescence.Previous studies have revealed that some WRKYs participate in the ROS clearance pathway; for example, transgenic rice plants overexpressing OsWRKY2 displayed increased ROS accumulation, whereas AtWRKY57 could confer ROS clearance 39,40 .Here, we found that overexpression of PtWRKY2 contributes to ROS elimination in Arabidopsis, which suggests that PtWRKY2 enhances thermotolerance, possibly by regulating ROS clearance.When plants encounter heat stress, a transcriptional network is activated to regulate the expression of thermoresponsive genes 41 .It has reported that HSF1s usually play a major role in these signaling networks 42 .In addition, DREB2A and bZIP28 are important heat shock response-related genes; mutant plants are usually hypersensitive to heat stress 43,44 .The upregulation of these genes (HSFA1A, DREB2A, and bZIP28) in PtWRKY2 transgenic plants suggested that PtWRKY2 may act as an important regulator in heat shock signal networks.Heat shock proteins (HSPs) are induced by high temperatures, and act as molecular chaperones that enhance thermotolerance 45 .A previous study revealed that WRKY family members could induce the expression of certain HSPs 46 , and we found HSP70 and HSP17.4 were both induced in the PtWRKY2 overexpressing lines, further suggesting that PtWRKY2 enhances thermotolerance, most likely by increasing the transcription of certain HSPs.Coinciding with the high activities of CAT, SOD, and POD, the expression of CAT1, SOD1 and POD34 was upregulated in the transgenic lines under heat stress, which is in line with the findings of Arabidopsis MEKK1 47 .Collectively, the transcriptional regulatory function of PtWRKY2 in response to heat stress was explored; however, the location of PtWRKY2 in the signaling networks remains unclear and requires further research.

Plant materials and growth conditions
P. ternata tubers (1 cm in diameter) were selected from the Experimental Farm of Huaibei Normal University (N 33°16′, E 116°23′, altitude: 340 m) and planted in potting soil.The potted plants were kept in a phytotron with a 16 h photoperiod and 35 µmol m −2 s −1 light intensity at 25 °C.When P. ternata reached the three-leaf stage, with a height of approximately 15 cm, its leaves, petioles, tubers, and roots were collected.To induce high-temperature stress, the three-leaf-stage seedlings were exposed to temperatures of 42 °C, while the photoperiod and light density remained unchanged.Whole plants were collected after 0, 4, 12, and 24 h of high-temperature stress.Each sample consisted of three plants, and three biological replicates were used for each treatment.
Col-0 background Arabidopsis was used as the wild-type (WT) line, and all Arabidopsis seeds were vernalized for 3 days and sown in a sterilized mixture with three parts nutrient soil and one part vermiculite.The seedlings were exposed to a 16 h light (50 µmol m −2 s −1 ) and 8 h dark cycle at 23 °C.The WRKY protein in P. ternata was searched in our previous transcriptome data 19 based on the sequences of WRKY conserved domains in Arabidopsis.Candidate WRKY genes of P. ternata were obtained by BLASTP analysis using a hidden Markov model of WRKY.The molecular weights (MWs) and isoelectric points (pIs) of the WRKY proteins were predicted using the ProtParam tool (https:// web.expasy.org/ protp aram/).The domains and conserved domains of the PtWRKY proteins were analyzed using Pfam (http:// pfam.xfam.org/) and MEME (http:// meme-suite.org/ index.html).Finally, TBtools software was used to produce the visualization diagram.The WRKY proteins from Arabidopsis and Oryza were downloaded from the NCBI database 48 , and the phylogenetic tree of WRKY proteins in P. ternata, Arabidopsis, and Oryza was established using MEGA7.0software and the Maximum Likelihood method, with the bootstrap setting as 1000.

Subcellular localization assay
The coding sequence of PtWRKY2 was amplified and then transformed into the vector pCAMBIA1302 for constructing a fusion plasmid, named 35S-PtWRKY2-GFP.The resulting 35S-PtWRKY2-GFP plasmid and empty pCAMBIA1302 control were transformed into tobacco epidermal cells refering to a previously published method 22 .GFP signals from the tobacco epidermal cells were captured under a fluorescence microscope (PA53 FS6, Motic, China).

Transcriptional activation activity assay
The full-length coding sequence of PtWRKY2 was cloned and fused to the GAL4 DNA-binding domain (DB) in the pGBKT7 vector to generate a recombinant vector, pGBKT7-PtWRKY2.The pGBKT7-53 plasmid and pGBKT7 empty vector were used as positive and negative controls, respectively.All plasmids were transformed into the Y2HGold yeast strain, which were subsequently cultured on SD/-Trp and SD/-Trp/-His/-Ade plates with or without X-α-gal.After 3-5 days' incubation of the yeast cells at 30 °C, the transactivation activity of PtWRKY2 was evaluated using a previously reported method 28 .

Dual-luciferase activity assay
A dual-luciferase activity assay of PtWRKY2 and W-box was performed according to a previously reported method 22 .Briefly, the PtWRKY2 coding sequence was amplified and transformed into the pGreenII62-SK vector to produce a fusion vector, PtWRKY2-62-SK.The three repeats of the W-box (TTG ACY ) and mW-box (TTT AAY ) were synthesized by oligonucleotide sequencing and cloned into the vector pGreenII-0800 to generate W-box-0800 and mW-box-0800, respectively.Using pGreenII-0800 as a control, the plasmid PtWRKY2-62-SK was co-transformed into N. benthamiana leaves with W-box-800, mW-box-800, and pGreenII-0800, respectively.After 72 h transfection, LUC signals were captured in the leaves using a multi-chemiluminescent imaging system (Tanon 5200, China).The activity of LUC/REN was determined using the Dual-LUC Assay Kit (Yeasen, China).

Plasmid construction and acquisition of transgenic plant material
The 1053 bp coding sequence of PtWRKY2 was amplified using its specific primers (Table S1), and introduced into the multiple cloning sites behind the CaMV 35S promoter in the pCAMBIA1301a vector; the result was an overexpression vector of the PtWRKY2 construct.The recombinant plasmid was transformed into Col-0 Arabidopsis via the Agrobacterium-mediated floral-dip method 49 .The T 3 homozygous lines were obtained for subsequent experiments.

High temperature tolerance analysis of transgenic plants
Newly harvested seeds from the WT and T 3 pure transgenic lines were selected for germination experiments.The seeds were sown on 1/2 MS solid media after surface sterilization, and treated with 4 °C for 3 days' vernalization.Thereafter, the seeds were kept at 23 °C for 1 day and subsequently exposed to a temperature of 42 °C for 0, 2, and 3 days.Next, seeds were cultured at 23 °C and captured at 14 days for germination rate calculations.For the root length experiments, the seeds were germinated on 1/2 MS solid media at 23 °C.When the root length reached approximately 0.5 cm, half of the germinated seeds were exposed to temperatures of 42 °C for 1 day, while the others were kept at 23 °C; the root length was measured after 10 days of growth.For the seedling survival rate assay, three-week-old WT and transgenic Arabidopsis plants were exposed to temperatures of 42 °C for 24 h, and subsequently transferred to a 23 °C environment for the survival rate calculation.Each experiment was repeated thrice.

Determination of ROS and antioxidant enzyme activity
Three-week-old WT and transgenic Arabidopsis plants were exposed to temperatures of 42 °C for 12 h, and the leaves were collected for reactive oxygen species (ROS) and antioxidant enzyme activity detection.The accumulation of hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 − ) in the leaves were analyzed via staining with

Figure 1 .
Figure 1.Phylogenetic analysis of the WRKY proteins of P. ternata, Oryza sativa, and Arabidopsis was performed using MEGA7.0, with a bootstrap setting of 1000.

Figure 2 .
Figure 2. Expression analysis of PtWRKY genes in P. ternata using RT-qPCR.(A) Expression profiles of PtWRKY genes in the roots, petioles, leaves, and tubers of two-month-old P. ternata plants.(B) Expression of PtWRKY genes in response to 42 °C heat stress.Values are presented as means ± SD (n = 3).Different letters indicate significant differences at P < 0.05.

Figure 4 .
Figure 4. Germination and root elongation analyses of wild-type (WT) and transgenic Arabidopsis under 42 °C heat stress.(A) PtWRKY2 expression levels in WT and three T 3 generation transgenic Arabidopsis plants based on RT-PCR.(B) Germination of WT and three PtWRKY2 transgenic lines on 1/2MS medium exposed to 42 °C heat stress for 0, 2, or 3 days.(C) Distribution diagram of WT and three PtWRKY2 transgenic lines on a plate.(D) Seed germination rates of WT and transgenic plants within 14 days.Root length (E) and statistical analysis (F) of WT and three transgenic Arabidopsis grown on 1/2MS medium exposed to 42 °C heat stress for 1 day and captured after 10 days.HS represents heat stress.OE1-2, OE6-3, and OE7-8 represent three independent pure transgenic lines.Different letters indicate significant differences at P < 0.05.

Figure 5 .
Figure 5. Heat tolerance of WT and transgenic Arabidopsis plants.Growth observation (A) and survival rate statistics (B) of WT and transgenic Arabidopsis under 42 °C heat stress for 1 day.Different letters indicate significant differences at P < 0.05.1DHS represents 1 d of heat stress.

Figure 6 .
Figure 6.Detection of reactive oxygen species (ROS) accumulation and antioxidant enzyme activity in WT and transgenic Arabidopsis plants under heat stress.Leaves of 3-week-old Arabidopsis plants grown under normal or heat stress conditions were sampled for (A) DAB and (B) nitro blue tetrazolium (NBT) staining and (C-E) catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities.Different letters indicate significant differences at P < 0.05.

Figure 7 .
Figure 7. Expression of heat shock-associated genes in WT and transgenic Arabidopsis plants under heat stress.* indicates significant differences compared to WT (P < 0.05).